Analytical validation of a second-generation immunoassay for the quantification of N-terminal pro-B-type natriuretic peptide in canine blood.

N-terminal pro-B-type natriuretic peptide (NT-proBNP) has been shown to have clinical utility as a biomarker in dogs with heart disease. There were several limitations associated with early diagnostic assay formats including a limited dynamic range and the need for protease inhibitors to maintain sample stability. A second-generation Cardiopet® proBNP enzyme-linked immunosorbent assay (IDEXX Laboratories Inc., Westbrook, Maine) was developed to address these limitations, and the present study reports the results of the analytical method validation for the second-generation assay. Coefficients of variation for intra-assay, interassay, and total precision based on 8 samples ranged from 3.9% to 8.9%, 2.0% to 5.0%, and 5.5% to 10.6%, respectively. Analytical sensitivity was established at 102 pmol/l. Accuracy averaged 102.0% based on the serial dilutions of 5 high-dose canine samples. Bilirubin, lipids, and hemoglobin had no effect on results. Reproducibility across 3 unique assay lots was excellent with an average coefficient of determination (r (2)) of 0.99 and slope of 1.03. Both ethylenediamine tetra-acetic acid plasma and serum gave equivalent results at time of blood draw (slope = 1.02, r (2) = 0.89; n = 51) but NT-proBNP was more stable in plasma at 25°C with median half-life measured at 244 hr and 136 hr for plasma and serum, respectively. Plasma is the preferred sample type and is considered stable up to 48 hr at room temperature whereas serum should be frozen or refrigerated when submitted for testing. Results of this study validate the second-generation canine Cardiopet proBNP assay for accurate and precise measurement of NT-proBNP in routine sample types from canine patients.